Proteases are proteolytic enzymes that catalyze the cleavage of peptide bonds in other proteins. The effect of such cleavage on protein molecules is diverse. In some instances proteolytic cleavage causes the cleaved protein to become inactive. In other instances proteolytic cleavage causes a once inactive protein to become activate. In yet other instances proteolytic cleavage is a mechanism whereby a single polypeptide precursor is cleaved into two or more individual polypeptides.
Proteolytic enzymes are grouped into families based on similar functions, active sites, amino acid sequences, and/or three-dimensional structures. Doolittle, Science, 214:149 (1981); and de Haen et al., J. Mol. Biol., 92:225 (1975). Examples of protease families are the cytochrome c family, the globins, mammalian serine proteases, and the cyclic nucleotide-dependent protein kinase family. Within proteolytic enzyme families are distinct subfamilies which usually share a similar type of physiological activity.
For instance, the common denominator of members in the serine protease family is a shared functional domain, i.e., the catalytic domain defined by amino acid residues Asp.sup.102, Ser.sup.195, and His.sup.57 of chymotrypsin. In addition, serine proteases sharing common physiological functions are categorized into subgroups. Examples of such subgroups are those containing serine proteases which function in the digestion, reproduction, and blood coagulation pathways.
Because proteases are involved in so many physiological processes, it is clinically useful to measure the level of a specific protease in body fluid samples. The measurement would indicate whether the specific protease is present at a level above or below that usually found in a body fluid sample, or may indicate whether a specific protease is present at at in situ, and may therefore provide a diagnostic index. Additionally, the measurement would provide insight into the fate of a specific protease administered to a patient in a therapeutic mode, or monitor the fate of a specific protease targeted by a therapeutic mode. A useful assay for a specific proteolytic enzyme should have the additional feature of indicating whether the specific proteolytic enzyme detected is "active" or capable of becoming activated.
An example of a protease for which measurement in a body fluid sample would be useful is activated protein C (APC), which is a member of the serine protease family subgroup involved in the blood coagulation pathway. Protein C (PC) is a zymogen, that is, it is inactive until converted into APC through interaction with thrombin, another serine protease active in the blood coagulation pathway. PC and APC are structurally different only in a dodecapeptide which is present at the amino-terminal end of PC and absence in APC. The 12 amino acid peptide is removed by proteolytic cleavage. The role of APC is to inactivate coagulation cofactors Va and VIIIa. Therefore, APC regulates thrombosis through its anti-thrombotic activity. In contrast to activation of PC by thrombin, APC is inactivated by the protein C inhibitors .alpha.-1-anti-trypsin, plasminogen activator inhibitor-1, .alpha.-2-antiplasmin, .alpha.-2-macroglobulin, and possibly other nonspecific proteases.
The level of APC and/or PC in a body fluid sample has medical relevance. For instance, the incidence of hereditary PC and protein S deficiency among thrombophilic patients [Gladson et al., Thromb. Haemost., 59:18-22 (1988)] is higher than in the normal population [Miletich et al., N. Engl. J. Med., 317:991-996 (1987)] and many patients have been described with heterozygous PC deficiency and familial thrombophilia [Griffin et al., J. Clin. Invest., 68:1370-1373 (1981); Horellou et al., Br. Med. J., 289:1285-1287 (1984); Bovill et al., Blood, 73:712-717 (1989)]. Complete deficiency of PC activity, whether inherited [Branson et al., Lancet, 2:1165-1168 (1983); Seligsohn et al., N. Engl. J. Med., 310:559-562 (1984)], experimental [Taylor et al., J. Clin. Invest., 79:918-925 (1987); Snow et al., Circulation, 82:III-769 (1990)], or acquired [Gruber et al., Thromb. Res., 42:579- 581 (1986); Mitchell et al., N. Engl. J. Med., 317:1638-1642 (1987)], represents a potentially fatal condition.
Thrombotic complications of PC deficiency can be controlled with PC or APC replacement therapy (Seligsohn et al., Taylor et al., and Snow et al., supra) or liver transplantation [Casella et al., Lancet, 1:435-437 (1988). The presence of measurable quantities of APC-inhibitor complexes in plasma samples from patients with intravascular coagulation indicates that APC is generated in vivo [Heeb et al., Blood, 73:455-461 (1989); Tabernero et al., Thromb. Haemost., 63:380-382 (1990)]. However, methods to detect unbound (free) APC in body samples have not been described except for assays measuring APC at elevated levels during APC infusion therapy of baboons. Gruber et al., Blood, 73:639-642 (1989). Thus, it is presently unknown if unbound APC is normally present in the vascular fluid, and if so, whether it can be measured.
Although it would be useful to detect the level of a functionally active protease with accuracy and sensitivity, including APC, in a body fluid sample, there are several limitations to achieving this goal. First, enzyme-activator interactions subsequent to sample collection will increase the activity of the protease to be measured. For example, PC present in a blood sample is activated by trace amounts of thrombin, thus increasing the level of APC measured to a value above that originally present, if at all, in a blood sample. Second, enzyme-inhibitor interactions subsequent to sample collection will decrease the activity of the protease to be measured. For example, APC which may be present in a blood sample is inactivated by any of a number of protease inhibitors, including those noted above, thus decreasing the level of APC to a value below that originally present, if at all, in a blood sample. Third, specificity of an assay depends on selective detection of the activity of only the protease desired to be detected. Thus, it is necessary to define a substrate that is highly specific for the protease to be detected. And, fourth, because proteases are present in the blood at very low concentrations, an assay to detect activity of a protease must be highly sensitive, without background interference due to factors such as those previously described.
Assays designed to detect APC in the blood have not overcome the obstacles delineated above and thus are not sufficiently sensitive to detect the activity of APC which might be present in a blood sample. For instance, Gruber et al., Blood, 73:639-642 (1989), describes an APC activity assay that measures the level of APC in the blood of baboons infused with APC. There, blood is drawn into benzamidine and citrate to block the serine protease activity in the blood sample and to inhibit complex formation between APC and its inhibitors. The treated blood then is contacted with the anti-PC monoclonal antibody C3 immobilized on microtiter plates, and the complex is washed to remove unbound blood constituents and benzamidine, a reversible inhibitor of APC. The washed complex is then contacted with a synthetic chromogenic substrate for APC and the amidolytic activity of APC is measured. The assay described in Gruber et al. supra was sensitive in the range of 0.005 to 5.0 ug/ml, a range suitable to detect the level of infused APC but too low to detect levels of APC which might be present in blood not infused with APC.
Another assay for APC activity described by Gruber et al., supra, is an APTT assay, i.e., an assay that measures the activated partial thromboplastin time (APTT) of plasma contacted with APC. Again, the data indicate that the APTT assay is useful in determining the level of APC in blood from a subject infused with APC, but is not sensitive enough to determine if APC is present in blood from a subject not infused with APC.
A functional assay for PC is described by Comp et al., Blood, 63:15-21 (1984). There, PC is activated in recalcified plasma by the thrombin-thrombomodulin complex, and contacted with immobilized anti-PC polyclonal antibodies. The activity of immobilized APC toward a synthetic substrate is measured. This assay provides an indication of the level of functional PC present in blood, i.e., PC that is capable of activation. The assay is not useful to determine the level of already activated PC, i.e., APC, that may be present in the blood.
The use of polyclonal antisera to measure PC activity also was described by Exner et al., J. Lab. Clin. Med., 107:405-411 (1986). PC was captured on immobilized antisera, activated, and then reacted with a chromogenic substrate. The assay does not detect the level of APC actually present in a blood sample.
Thus, while functional assays to detect PC in the blood and to detect APC in blood infused with APC are known, there remains a need for sensitive assays to accurately detect whether an active protease such as APC is present in a body fluid sample and, if present, to quantitate its presence.